Quantification of synaptic protein co-localization a. Excitatory synaptic protein expression in segments of dendrites from neurons grown for 7, 14 or 21 DIV on either WT astrocytes (left panel) and on Fmr1 -/- astrocytes (right panel). Each set of three images represents the same 50 μm segment visualized with immunofluorescence directed at pre- and post-synaptic proteins (top, merge), pre-synaptic synapsin (green, middle), and post-synaptic PSD-95 (red, bottom). b. From similar 50 μm segments, the number of synapses (i.e., instances of co-localized puncta) per 50 μm were quantified. The number of days in culture (DIV) are indicated along the x-axis. The solid bars are data from neurons grown on WT astrocytes. The white bars are data from neurons grown on Fmr1-/- astrocytes. Data are presented as mean ± s.e.m., from 300 segments from three independent experiments. ***, significant at P < 0.001, Mann Whitney U tests (two-tailed).